Field of the Invention
The present invention relates to a hybrid image generating method of an optical system, especially to a pseudo H&E image producing method and an optical system using the method.
Description of the Related Art
In conventional tumor surgery, it takes a lot of time for a pathologist to examine a frozen section to determine if a tumor is cleanly removed. However, during the process of preparing a frozen section of a pathological sample, if the pathological sample contains much water, a formed crystal ice structure will cause a damage to the tissue structure of the pathological sample; and if the pathological sample contains much fat, the fat will remain unfrozen at temperatures around −20° C. where the other tissues are already frozen, and is therefore easy to slip off from the frozen section to cause an artifact to the frozen section. Besides, frozen cells are generally not easily stained. Due to the facts disclosed above, the artifact, which does not exist in fresh tissues of the pathological sample, will be present in the image of the frozen section of the pathological sample. To cope with this problem, optical inspection instrument has been adopted to provide a real time pseudo H&E image of a sliced sample for medical personnel to inspect, and the pseudo H&E image is derived by a non-destructive optical sectioning scheme without the need of slicing a frozen sample.
General optical sectioning method performs a linear combination of a plurality of grayscale images of a pathological sample to generate a black-background RGB image, so that when the black-background RGB image is viewed by the eyes of a person, the person will sense as if a color image is present in a dark space. The plurality of grayscale images are generated by performing a plurality of different image producing methods on the pathological sample, the color of each pixel of the color image is determined by a combination of three primary colors (red, green, blue), and the intensity values of the three primary colors of each pixel are determined by three linear combinations of corresponding pixels of the plurality of grayscale images. Besides, conventional image producing scheme, such as satellite image scheme, X-ray image scheme, or electronic microscopic image scheme, can only present image of grayscale format due to single image intensity distribution. If the image can be displayed in color format, not only readability and distinguishability of the image can be improved, but also more information can be provided with the image.
Besides, as far as color contrast is concerned, red and green have the highest degree of contrast. However, when a person has viewed a lot of red-and-green images with black background or has viewed a red-and-green image with black background for a long time, his/her eyes will feel uncomfortable and fatigued. As a result, the format of red-and-green image with black background is not suitable for the image of a pathological sample, because a pathologist may have to inspect a lot of images of pathological samples during a work day. At present time, pathological image is generally generated by firstly staining a pathological sample section (paraffin section or frozen section) with Hematoxylin and Eosin, and then executing an image producing procedure to generate a tender white-background image, where the Hematoxylin and Eosin will paint nuclei and cytoplasm with blue-purple color and pink color respectively, and as different parts in the cytoplasm respond differently in sticking to the Eosin and by which different shades of pink color will be generated correspondingly, the H&E staining color image can therefore provide more detailed pathological information. Therefore, if the commonly used black-background RGB image can be replaced with white-background RGB image like H&E image, it will be very helpful to a pathologist who needs to inspect a lot amount of images. However, as traditional pathological image producing method not only has an artifact issue, but also requires a long time, it is therefore not suitable for providing a timely pathological image needed in a surgery.
U.S. Pat. No. 8,269,827B2 “System and methods for mapping fluorescent images into a bright field color space” discloses a method of using fluorescent images to generate a pseudo H&E image, including: deriving two or more fluorescent images of a specified area of a pathological sample; and using mapping parameters to transfer the fluorescent images into a bright field color space to generate a bright-field image.
However, as the U.S. Pat. No. 8,269,827B2 uses fluorescent images to generate a pseudo H&E image, it has disadvantages as follows: (1) the color contrast of the pseudo H&E image is not good enough due to a fact that the internal structure of a pathological sample cannot be uniformly stained with a fluorescent agent; (2) the fluorescent agents used for generating the fluorescent images will cause damage to the pathological sample; and (3) it takes a lot of time to stain the fluorescent agents onto the pathological sample. Therefore, there is a need of a novel method for producing a pseudo H&E image in the field.